Project Summary/Abstract Background. Critical limb ischemia (CLI), a devastating peripheral arterial disease, affects patients with type 2 diabetes (T2D) and obesity, and is prevalent in minorities. Half of cases require amputation, leading to up to 40% mortality and worse prognosis in minorities. There is no effective medical treatment. Several stem cells are promising in clinical trials, but there are three concerns: a) studies based mainly on their angiogenic capacity, but not on their efficacy in skeletal muscle regeneration or neural repair; b) stem cell autografts from the T2D patient may be severely impaired by long term exposure to the T2D milieu; and c) the ischemic/diabetic tissue impact on the implanted stem cells is not targeted. We have shown: a) at short periods muscle derived stem cells (MDSC) stimulate early myogenesis in the muscle of the db/db mouse with CLI, but this is accompanied by myostatin overexpression and fibrosis that interfere with myofiber formation; b) the T2D milieu damages MDSC and impairs their repair capacity by in another diabetic complication, imprinting their transcriptional signature; c) pioglitazone at low doses prevents inflammation and fibrosis independently from glycemic control, and myostatin inhibitors stimulate muscle mass and are antifibrotic and proangiogenic. Overall goal: to optimize in a T2D mouse model the stem cell therapy of CLI, comparing MDSC with a stem cell currently in clinical trials for CLI, adipose derived stem cells (ADSC). This is in order to: a) stimulate myogenesis and regenerate striated myofibers, in addition to neoangiogenesis; and b) define and counteract the impairment of stem cell repair capacity upon long exposure to the T2D milieu, by pharmacological modulation with pioglitazone, a PPAR? agonist, and/or shRNA against myostatin as inhibitor of this negative regulator of muscle mass and pro-lipofibrotic effector, and study their mechanism and impact on the stem cells transcriptional signatures. Specific Aim 1. To determine in the T2D mouse model of CLI: 1) the repair efficacy of MDSC and ADSC on the ischemic/diabetic muscle, and the impact of their prior exposure to the T2D milieu on efficacy and underlying transcriptional signatures; and 2) whether pharmacological modulation with pioglitazone and/or a myostatin inhibitor stimulates stem cells repair and counteracts the T2D-induced damage. Specific Aim 2. To characterize in vitro: a) the roles of hyperglycemia and/or dyslipidemia in the T2D-induced stem cell damage; b) a preliminary insight on the mechanism of pioglitazone and myostatin inhibition effects on survival and differentiation of stem cells, and on their transcriptional signatures affected by treatments. Impact/innovation: High translational impact of a very novel approach aiming to improve the efficacy of stem cells targeting myofiber repair, through concurrent pharmacological modulation with a new modality for a widely clinically used drug and a promising ancillary approach, to define if and how the T2DM milieu damages them, and to counteract this damage through the drug combinations.